1. Field of the Invention
The present invention relates to a analyzing method and an analyzing apparatus for analyzing an observed target region in a biological specimen, emitting a feeble light, with a suitable resolution. The present invention incorporates herein the content of PCT/JP2006/319589, which is an international patent application filed by the present applicant, by applying the method and the apparatus disclosed in PCT/JP2006/319589 to the present application.
The present invention relates to a biological specimen imaging method and a biological specimen imaging apparatus for imaging an observed target region in a biological specimen that emits a feeble light.
The present invention relates to a method for detecting a biological activity in a biological specimen, such as a cell or a tissue, for a long time or continuously, without deteriorating the activity as much as possible.
The present invention includes software for an automated apparatus executed by the method.
2. Description of the Related Art
[I] In recent years, an imaging technique of a biological sample utilizing fluorescence has taken a great role for a research of a bioscience. A specific protein is marked, and light emission is utilized, whereby various life phenomena happening inside or outside a cell can be observed. Further, dynamic actions of various life phenomena can be known in real time. In recent days, in particular, the use of a fluorescent protein such as GFP (Green Fluorescent Protein) makes it possible to stably and easily realize an imaging of a structure in a cell, so that various life phenomena has been steadily unraveled.
Further, a luminescence-related gene that expresses a bioluminescent protein (specifically, luciferase, aequolin, or the like) has frequently been utilized for various function analyses in a cell (specifically, a luminescence-related gene has frequently been utilized as a reporter molecule of a protein expression). In performing the function analyses described above, it is extremely significant to describe a clear image by bringing a lens into focus on a specific region in a cell, or to efficiently receive light emitted from a specific bioluminescent protein transduced to a cell. However, since an intensity of light of self-luminescence from a biological specimen is generally extremely feebler the luminescence from the biological specimen cannot directly be confirmed with naked eyes in most cases. Even when an optical element (e.g., detection lens, etc.) is adjusted to a specimen emitting a feeble light, it is naturally difficult to bring a lens into focus on the specific region in the specimen with naked eyes.
In view of this, various methods for focusing light on a specimen in a specimen container have been disclosed. However, all of the methods are applicable to the case in which the light from a specimen can be confirmed (visually) with naked eyes. For example, JP-T-2002-541430 and JP-T-2002-542480 disclose a method in which a position of a bottom surface of a specimen container is detected, and light is focused on a specimen in the specimen container based on the detected positional information. In JP-T-2002-541430, light is irradiated to the bottom surface of the specimen container through an objective lens, the intensity of the mirror-reflected light from the bottom surface of the specimen container is sequentially detected while moving the light irradiation position vertically, the position of the bottom surface of the specimen container is detected based on the detected intensity of light, the position of the specimen in the specimen container is estimated based upon the detected positional information, and light is focused on the estimated position. In JP-T-2002-542480, light is diagonally irradiated to the bottom surface of the specimen container from below through an objective lens, a deviation amount of the mirror-reflected light from the bottom surface of the specimen container on an XY plane is detected by a photodetector, the position of the bottom surface of the specimen container on an optical axis is detected based upon the detected deviation amount, the position of the specimen in the specimen container is estimated based upon the detected positional information, and light is focused on the estimated position. Thus, the focal position of the objective lens can be focused on the specimen in the specimen container.
JP-A-2004-354650 and JP-A-2005-173288 disclose a method in which, with the use of a microscope for observing a phase object such as a cell with a bright field, the position of an objective lens is shifted in the forward or rearward direction from the general focusing position to be fixed to thereby obtain an observation image with a high contrast by defocus, in order to observe a cell. With this method, the observation image of the cell, which is the phase object, can be obtained with a high contrast.
[II] In the research of a biological science or a medical science, a technique of detecting a biological activity of a biological specimen such as a cell with a reporter assay has widely been utilized. The use of the reporter assay can make various biological activities, which cannot visually be examined, visible. In a conventional clinical examination, only biological materials (nucleic acid, blood, hormone, protein, etc.), which are to be examined, are isolated from the biological specimen by various isolation methods, and the amount or activity of the isolated biological material is reacted with a reagent. However, in a living body, it is the interaction among varied biological materials that exhibits the true biological activity. When a medical agent is studied or developed, the decisive condition for the agent is that the agent is most effectively acted on a biological activity in a living biological specimen. In the reporter assay targeted for a living biological specimen, it is more required that images of a biological specimen and a biological material to be examined are formed for observing a dynamic change at the inside or outside of the biological specimen over time.
Specifically, in a research field utilizing an observation using luminescence (bioluminescence, chemiluminescence) or fluorescence as a reporter substance, a time-lapse or an image-capture of a moving image is demanded in order to catch a dynamic functional expression of a protein molecule in a specimen. Under the present condition, a dynamic change in an image is observed by imaging a fluorescent specimen as a subject (e.g., a moving image of one protein molecule is observed by utilizing fluorescence). When a fluorescent specimen is imaged, it is difficult to take, over time, a stable image that can be used for a quantitative evaluation, because the amount of light emitted from the fluorescent specimen tends to decrease with the lapse of time due to the continuous irradiation of excited light, but a clear image, i.e., an image having high spatial resolution, can be imaged in a short exposure time. On the other hand, in the observation of a dynamic change over time according to an image of a luminescent specimen, the luminescent specimen is observed with the use of a CCD camera having an image intensifier mounted thereon, because the emission from the luminescent specimen is extremely feeble. In the case of imaging the luminescent specimen, a stable image that can be used for a quantitative evaluation can be taken over time, because there is no need to irradiate excited light.
The emission amount from a luminescent specimen is measured in the observation of a luminescent specimen. For example, in the observation of a cell to which luciferase gene is transduced, the light emission amount from the cell due to the luciferase activity is measured in order to examine the strength of the expression of the luciferase gene (specifically, the expression amount). The light emission amount from a cell according to a luciferase activity is measured as follows. Specifically, a cell lysate in which a cell is lysed and a substrate solution containing luciferin, ATP, magnesium, etc, are reacted, and then, the light emission amount from the cell lysate reacted with the substrate solution is quantified by means of a luminometer using a photomultiplier tube. In other words, the light emission amount is measured after the cell is lysed. Thus, the expression amount of the luciferase gene at a certain point can be measured as an average of the whole cell. The methods for transducing a luminescent gene such as a luciferase gene into a cell as a reporter gene include, for example, a calcium phosphate method, lipofection method, electroporation method, etc. Each method is used according to the purpose or a type of a cell. When the strength of the expression of the luciferase gene is examined, with the light emission amount from the cell according to the luciferase activity defined as an index, in a cell into which the luciferase gene is transduced as a reporter gene, a target DNA fragment is coupled to the upstream side or the downstream side of the luciferase gene that is transduced into the cell so as to examine the influence given by the DNA fragment to the transcription of the luciferase gene, and further, a gene, such as a transcription factor, that is considered to affect the transcription of the luciferase gene transduced to the cell is coupled to an expression vector so as to co-express the gene with the luciferase gene, whereby the influence given by the gene product of the gene to the expression of the luciferase gene can be examined.
In order to catch the amount of the expression of a luminescent gene with the lapse of time, it is necessary to measure a light emission amount from a living cell over time. The light emission amount from a living cell is measured over time as follows. Specifically, a function of a luminometer is provided to an incubator that cultivates a cell, and then, the light emission amount from all of the cultivated cell populations is quantified by the luminometer for every predetermined time. Thus, the expression rhythm with a constant periodicity can be measured, whereby the change in the amount of the expression of the luminescent gene in all the cells can be caught over time. On the other hand, when the expression of a luminescent gene is transient, the amounts of the expression in individual cells greatly vary For example, even in a cloned cultured cell such as HeLa cell, the response to an agent via a receptor on the surface of the cell membrane might vary in the individual cells. Specifically, although the response as the whole cell may not be detected, several cells might make a response. From this, it is important to measure the light emission amount over time from not the whole cell but the individual cell, in case where the expression of the luminescent gene is transient. Because light emission from each cell is significantly feeble, the measurement over time of the light emission amount from an individual living cell by means of a microscope is carried out by exposing the cell with a cooled CCD camera with a temperature level of a liquid nitrogen for a long timer or by using a CCD camera provided with an image intensifier and a photon counting apparatus. Thus, a change over time in an amount of expression of a luminescent gene in an individual living cell can be obtained.
In the description above, the analyzing method and apparatus for the expression of a gene, using a fluorescent protein as a reporter gene is disclosed in JP-T-2004-500576, for example. The analyzing method and apparatus for the expression of a gene with the use of a luminometer according to bioluminescence is disclosed in JP-A-2005-118050, for example.